This proposal describes how specific enzymes control the transfer of electrons and activation of molecular oxygen, while minimizing oxidative damage. This is central to cell development, health and survival. This project includes studies of enzyme reaction mechanisms, protein structure-function relationships, protein-protein interactions, protein post-translational modification, and mechanisms of long range biological electron transfer. Kinetic, biochemical, spectroscopic and structural studies together with site-directed mutagenesis will be used in these studies. This proposal focuses on the mechanism of biosynthesis ofthe protein-derived cofactor, tryptophan tryptophylquinone (TTQ), and the structure and function of a novel di-heme enzyme MauG which catalyzes the oxygenation and cross-linking of specific tryptophan residues during TTQ biogenesis in methylamine dehydrogenase (MADH). The substrate for MauG is a 119-kDa precursor protein of MADH with mono-hydroxylated Trp57 and no cross-link. MauG catalyzes the 6-electron oxidation of the substrate that results in the second oxygenation of Trp57, cross-linking of Trp57 and Trp108, and oxidation of the quinol product of the first two reactions to form oxidized TTQ. These studies will describe a new biological mechanism for oxygen activation and factors that make specific amino acid residues in proteins susceptible to oxidative modification.The results will provide insight for development of strategies to introduce novel catalytic sites into proteins and manipulate the functions of enzyme-bound hemes, as well as provide clues as to how one might mitigate naturally occurring oxidative damage to proteins. Ongoing mechanistic studies of biological electron transfer (ET) in the MADH-amicyanin-cytochrome c-551 i protein complex will be extended and ET studies will be initiated with MauG. Defining mechanisms of long range ET reactions will enhance our understanding of the fundamental processes of respiration and intermediary metabolism at the molecular level. A fundamental understanding of the mechanisms of control of biological ET reactions will provide insight into how defective protein ET leads to production of reactive oxygen species and free radicals both of which are associated with many disease states, oxidative stress and aging. RELEVANCE (See instructions): Reactive oxygen species and free radicals produced as by-products of biological electron transfer and oxygen metabolism damage cell components that cause many disease states, oxidative stress and aging. However, free radicals and reactive oxygen species are also used productively in biosynthetic processes. These studies describe how specific enzymes control the transfer of electrons and activate oxygen, while minimizing oxidative damage.